To date, ovarian cancer remains the number one killer of women with gynecologic malignant hyperplasia. Approximately 75% of women diagnosed with such cancers are already at the high-stage (III and IV) of the disease at their initial diagnosis. During the past 20 years, neither diagnosis nor five year survival have greatly improved for these patients. This is substantially due to the high percentage of high-stage initial detections of the disease. Therefore, the challenge remains to develop new markers to improve early diagnosis, and reduce the percentage of high-stage initial diagnoses.
A good tumor marker useful as an indicator of early disease is needed. Extra-cellular proteases have already been implicated in the growth, spread and metastatic progression of many cancers, thereby implying that some extracellular proteases may be candidates for marker of neoplastic development. This is in part due to the ability of malignant cells not only to grow in situ, but to dissociate from the primary tumor and to invade new surfaces. The ability to disengage from one tissue and re-engage the surface of another tissue is what provides for the morbidity and mortality associated with this disease.
In order for malignant cells to grow, spread or metastasize, they must have the capacity to invade local host tissue, dissociate or shed from the primary tumor, and for metastasis to occur, enter and survive in the bloodstream, implant by invasion into the surface of the target organ and establish an environment conducive for new colony growth (including the induction of angiogenic and growth factors). During this progression, natural tissue barriers have to be degraded including basement membranes and connective tissue. These barriers include collagen, laminin, proteoglycans and extracellular matrix glycoproteins including fibronectin.
Degradation of these natural barriers, both surrounding the primary tumor and at sites of metasttic invasion is believed to be brought about by the action of a matrix of extracellular protease. Proteases have been classified into four families: serine proteases, metallo-proteases, aspartic proteases and cysteine proteases. Many proteases have been shown to be involved in the human disease process and these enzymes are targets for the development of inhibitors as new therapeutic agents.
Certain individual proteases have already been shown to be induced and over expressed in a diverse group of cancers, and as such, are potential candidates for markers useful for early diagnosis and possibly therapeutic intervention. A group of examples are listed below. The list of enzymes encompasses members of the metallo-proteases, serine proteases, and cysteine proteases as shown in Table 1.
TABLE 1 Protease Expression in Various Cancers Gastric Brain Breast Ovarian Serine Proteases uPA uPA NES-1 NES-1 PAI-1 PAI-1 uPA uPA tPA PAI-2 Cysteine Proteases Cathepsin B Cathepsin L Cathepsin B Cathepsin B Cathepsin L Cathepsin L Cathepsin L Metallo-proteases Matrilysin* Matrilysin Stromelysin-3 MMP-2 Collagenase* Stromelysin MMP-8 Stromelysin-1* Gelatinase BMMP-9 Gelatinase A uPA-Urokinase-type plasminogen activator, tPA - Tissue-type plasminogen activator, PAI-I - Plasminogen # activator 0 inhibitors, PAI-2 - Plasminogen activator inhibitors, NES-1 - Normal epithelial cell-specific-1, # MMP - Matrix P metallo-protease. *These metallo-proteases are over expressed in gastrointestinal ulcers.
Significantly there is a good body of evidence supporting the down regulation or inhibition of individual proteases and reduction in invasive capacity or malignancy. In work by Clark et al. inhibition of in vitro growth of human small cell lung cancer was demonstrated using a general serine protease inhibitor. More recently, Torres-Rosedo et al., Proc. Natl. Acad. Sci. USA, 90, 7181-7185 (1993). demonstrated an inhibition of tumor cell growth of hepatoma cells using specific antisense inhibitors for the serine protease hepsin gene. Metastatic potential has also been shown to be reduced in a mouse model with melanoma cells by using a synthetic inhibitor (batimastat) of metallo-protease. Powell, et al. Cancer Research, 53, 417-422 (1993), presented evidence to confirm that the expression of extracellular proteases in relatively non-invasive tumor cells enhances their malignant progression using a tumor-genic but non-metastatic prostate cell line. Specifically, they demonstrated enhanced metastasis after introducing and expressing the PUMP-1 metallo-protease gene. There is also a body of data to support the notion that expression of cell surface proteases on relatively non-metastatic cell types increases the invasive potential of such cells.